Non-coding RNAs (ncRNAs) are a class of epigenetic modifiers with growing roles in transcriptional regulation. Of particular interest is a lineage-restricted class of unspliced ncRNAs, defined as enhancer RNAs (eRNAs), which are bidirectionally transcribed from a minority of highly-active enhancers. Enhancers are cis- regulatory elements (CREs) with critical role(s) in lineage-specific transcription. While the function of enhancers in driving cell-type specific gene expression has been known for decades, only a few studies have described a role of eRNAs in transcriptional regulation. This proposal focuses on a novel role of eRNAs in sub- nuclear localization, an important category of epigenetic regulation. No study has examined a function of eRNAs in altering sub-nuclear localization by excluding their linked enhancer from the nuclear lamina (NL), a movement associated with transcriptional activation of genes. Embryonic stem cells (ESCs) provide an ideal model to delineate the mechanism of eRNAs in transcription and sub-nuclear localization, as they can be differentiated into virtually any cell lineage. This study will show a critical roleof eRNAs at transcription factor (TF) loci as pluripotent ESCs enter the myeloid lineage. Decommissioning of pluripotent TF enhancers and activation of myeloid TF enhancers, as indicated by loss and gain of eRNA production respectively, is necessary for ESCs to undergo myelopoiesis. A better understanding of the many levels of epigenetic regulation during lineage commitment is crucial to the advancement of personalized medicine by facilitating our ability to differentiate patient induced pluripotent stem cells into any lineage for cellular therapies. This proposal focuses on eRNAs produced at well-characterized loci for the pluripotent TF Nanog and essential myeloid TFs PU.1 and C/EBPa. To decipher a function of eRNAs, RNAi will be used to deplete cell- type specific enhancer transcripts. Establishing a paradigm for eRNAs in enhancement of transcription and sub-nuclear localization in ESCs, permits similar studies to be performed in myeloid cells. While there has been some work studying various classes of ncRNAs (including eRNAs) during erythropoiesis, no one has investigated eRNAs during myelopoiesis. An in vitro role of myeloid eRNAs in determining cell fate can be assessed by differentiating eRNA depleted ESCs into blood cells. Accordingly, an in vivo role can be tested by depleting myeloid eRNAs in bone marrow cells and transplanting into irradiated syngeneic mice. In sum, while a few studies have shown a role of eRNAs in transcriptional regulation, this proposal will define a new function of eRNAs in sub-nuclear localization, a type of epigenetic regulation controlling cell-type specific gene expression. Moreover, no one has studied the effect of depleting ESC or myeloid eRNAs and examined the effects in vitro and in vivo.